1. Field of the Invention
The human serum contains at least three known binders of Vitamin B-12, namely the three transcobalamins designated as TC-I, TC-II, and TC-III. TC-I and TC-III are derived from granulocytes and both are alpha-globulins with a similar molecular weight, of about 120,000. They have a different electrical charge and hence differ in their electrophoretic mobility. TC-II is a beta-globulin of molecular weight of about 38,000 and it is derived mainly from the liver. The physiological functions of the three transcobalamins are not fully understood, but it is known that endogenous Vitamin B-12 is bound mainly to TC-I (about 85%), and TC-II binds about 15% of endogenous B-12 while TC-III seems to bind Vitamin B-12 only in vitro. Since TC-II binds small quantities of endogenous B-12 while it takes up the main part of Vitamin B-12 added to the serum in vitro, most of the unsaturated B-12 binding sites are located on TC-II (unsaturated B-12 binding capacity, UBBC). Vitamin B-12 is bound in the serum to the transcobalamins in a 1:1 molar ratio.
It is well known that certain pathological conditions are associated with significant specific changes in the level of the three transcobalamins in serum and that the determination of the Vitamin B-12 binding capacity of each of the three transcobalamins is an important tool in medical diagnosis. Amongst others, the quantitative determination of the B-12 binding capacity of the three transcobalamins is of value in the effective screening of certain malignant diseases and also in the monitoring of the treatment of these diseases. Amongst others, the determination of the three transcobalamins is of value in:
A. Diagnosis, evaluation of treatment and monitoring of the course of myeloproliferative diseases [CML (chronic myelocytic leukemia), APL (acute promyeolocytic leukemia), polycythemia vera.] PA1 B. Differentiation of leukemoid reactions and conditions manifested by non-leukemic leukocytosis. PA1 C. Recognition of rapid malignant cell proliferation in lymphoma, sarcoma, Hodgkins Disease, actue leukemia, etc. PA1 D. Evaluation of therapy and monitoring the course of malignant diseases (remission and relapse) such as sarcomas, actue leukemias, Hodgkins Disease, lymphomas etc. PA1 E. Diagnosis and recognition of hepato-cellular damage.
The quantitative determination of B-12 binding capacity of the three transcobalamins may also be of value in the recognition, differentiation and monitoring of various other disorders.
2. Brief Description of the Prior Art
The three transcobalamins present in human serum are difficult to separate and their quantitative determination is both complicated and time-consuming. The main problem is the similarity of electrophoretic properties of TC-II and TC-III and their similar behavior on DEAE-cellulose separation.
The prior art techniques for separating the transcobalamins are characterized by the methods described in the following references: Blood 33:899(1969), Br. J. Haematol. 22:33 and 53(1972), J. Lab. Clin. Med. 73:60(1969), Am. J. Clin. Pathol. 62:367(1974), Blood 31:518(1968), Blood 25:875(1965), Fed. Proc. 33:715(1974), blood 45:281 and 287(1975), J. Lab. Clin. Med. 75:673(1970), Proc. Soc. Exp. Biol. Med. 147:377 (1974), and J. Biol. Chem. 250:7700(1975). These prior art techniques are cumbersome research techniques and some are even known to give inconsistent results. Some require days to complete and all have to be considered as confined to use in the research laboratory.
An object of the present invention is to provide a process and test kit for transcobalamin determination which is applicable to routine use in the clinical laboratory. The present inventors, in FEBS Letters 44:71(1974), described an advantageous technique for the separate determination of TC-I and TC-II, but made no suggestion as to the determination of TC-III as well, which substance has properties similar to TC-I.